Purification of a recombinantly produced transmembrane protein (gp41) of HIV I.
The transmembrane protein gp41, a component of the viral envelope of HIV I, and its analogue gp36 of HIV II are important antigens for the sensitive and specific detection of anti-HIV antibodies. The immunodominant region of the protein gp41, which reacts with 100% of sera of infected persons, was produced by gene technological means in Escherichia coli. The protein accumulates in the form of insoluble inclusion bodies in the bacterial cell. Purification strategies for this aggregated material depend mainly on the isolation of these "inclusion bodies" and subsequent washing procedures. Growth conditions of the recombinant E. coli cells and the method of the cell disruption are important for the efficiency of purification and the recovery of the antigen. Owing to the insolubility of the expressed antigen, a significant concentration of recombinant gp41 was possible by extracting the soluble cell components. For this purpose, mild detergent solutions and low-molarity chaotropic buffer solutions were used. After final solubilization in 8 M urea buffer at pH 12.5, further chromatographic purification steps followed. The reduction of disulphide bridges with beta-mercaptoethanol or dithiothreitol was important. Gel filtration on a Sephacryl S-200 or Superose 12 column and/or ion-exchange chromatography on a DEAE-Sepharose Fast Flow or Mono Q HR (5/5) column finally resulted in the desired purity of the antigen.
Soutschek
E
E
Mikrogen GmbH, München 2 F.R.G.
Höflacher
B
B
Motz
M
M
D015153:Blotting, Western; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000032:analysis / Q000378:metabolism; D005822:Genetic Vectors; D015700:HIV Envelope Protein gp41 / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification*
D015700:HIV Envelope Protein gp41; D011994:Recombinant Proteins